Electronic supplementary material

The online version of this article (doi:10.​1007/​s12026-017-8936-9) contains supplementary material, which is available to authorized users.

Daniela Sauma and Natalia Crisóstomo contributed equally to this work.

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by increased autoantibody production that leads to multiple tissue injuries. Dendritic cells (DCs) are important orchestrators of immune responses and key components in fine-tuning the balance between tolerance and immunity. However, their role in autoimmune disorders such as SLE remains uncertain. We analyzed the contribution of DCs in triggering SLE by adoptively transferring splenic DCs from aged autoimmune [NZB×NZW]F1 (BWF1) mice to young healthy BWF1 mice. We observed that the transfer of DCs from autoimmune mice to pre-autoimmune mice induced high autoantibody titers in the serum of recipient mice. Moreover, autoimmune DCs from aged BWF1 mice were crucial for the expansion and differentiation of plasmablasts and CD5+ B cells or B1-like cells in the peripheral blood, and spleen of recipient BWF1 mice, a phenomenon that is observed in autoimmune BWF1 mice. On the other hand, DCs from aged BWF1 mice participated in the expansion and differentiation of DCs and IFN-γ-producing T cells. These results reveal that DCs from autoimmune BWF1 mice exhibit functional and phenotypic characteristics that allow them to trigger B cell hyperactivation, as well as DC and T cell expansion and differentiation, thereby promoting an exacerbated humoral response in lupus-prone mice.

Suppl. Fig. 1 Characterization of CD11c+ cellular populations used for the adoptive transfer and co-culture experiments. a FACS analysis of sorted splenic CD11c+ cells obtained from aged [NZWxBALB/c]F1 control or aged autoimmune BWF1 mice after the depletion of B and T cells. The graphs on the left show the percentage of NK1.1− and NK1.1+ cells over total live CD11c+ cells. Graphs on the right show the percentage of pDC (CD11cintB220+) and cDC (CD11chiB220−) cells gated on live NK1.1− cells (numbers represent the percentage of cells in each gate). b Comparative expression of CD11b, CD49b, I-Ad and PDCA1 in pDCs (top), cDCs (middle) and NK1.1+ (bottom) cell subsets defined previously on a for control (dashed line histograms) or autoimmune CD11c+ cells (continuous line histograms). Background staining is represented as gray histograms. (DOCX 426 kb)

Suppl. Fig. 2 Transfer of autoimmune DCs to [NZWxBALB/c] F1 control mice does not induce anti-dsDNA autoantibodies. a Splenic DCs from aged [NZWxBALB/c]F1 control (control DCs) or aged BWF1 mice (autoimmune DCs) were sorted and injected i.v. (4 × 106/mouse) into groups of young control [NZWxBALB/c]F1 mice at days 0 and 20 (black arrows). Serum was obtained every eight or ten days after the first injection over the course of 75 days and tested for anti-dsDNA auto-antibodies by standard ELISA. White circles: sera from young [NZWxBALB/c]F1 mice treated with control DCs (n = 2); black circles: sera from young [NZWxBALB/c]F1 mice treated with autoimmune DCs (n = 3); shaded area: sera from young, untreated BWF1 mice. b Splenic pDCs and cDCs from aged BWF1 mice (autoimmune DCs) were sorted and injected i.v. (0.6 × 106/mouse) into young BWF1 mice. Serum was obtained every five days over the course of 25 days and tested for anti-dsDNA autoantibodies by standard ELISA. Black circles: sera from young [NZWxBALB/c]F1 mice treated with autoimmune pDCs (n = 1); black squares: sera from young [NZWxBALB/c]F1 mice treated with autoimmune cDCs (n = 1); white triangles: sera from young, untreated BWF1 mice. (DOCX 305 kb)

Suppl. Fig. 3 Effect of the transfer of autoimmune DCs or control DCs on the absolute number of naïve and memory B cells. a Absolute numbers of splenic IgM+IgD+ naïve and b IgM+IgD− memory B cells, of young BWF1 mice 60 days post-injection of control or autoimmune DCs. The data in the graphs are presented as the mean ± S.E.M. (n = 4 mice per group) (two-tailed Mann-Whitney test). (DOCX 92 kb)

Suppl. Fig. 4 Effect of the transfer of autoimmune DCs or control DCs on the absolute number of CD19+CD138+ plasmablast cells and CD19−CD138+ plasma cells 60 days post-injection of autoimmune DCs or control DCs. The data in the graphs are presented as the mean ± S.E.M. (n = 4 mice per group) (two-tailed Mann-Whitney test). (DOCX 64 kb)

Suppl. Fig. 6 B10 and Breg subsets are highly expanded in the spleens of young BWF1 mice injected with autoimmune DCs. a Representative FACS analysis of IL-10 intracellular staining of LPS/PMA/ionomycin-stimulated B cells from the spleen of young BWF1 mice injected with control or autoimmune DCs (numbers represent the percentage of cells in each gate). The graph on the right shows the percentage of IL-10+ cells within a CD19+ gate. b Representative FACS analysis of B cells from the spleens of young BWF1 mice injected with control or autoimmune DCs (numbers represent the percentage of events in each gate). The graph on the right shows the percentage of CD1dhiCD5+ cells within a CD19+ gate. The data in the bar graphs are presented as the mean ± S.E.M. (n = 4 mice per group). *p < 0.05 (two-tailed Mann-Whitney test). (DOCX 240 kb)